The present invention relates to a new class of nucleoside analogues and their therapeutic use in the prophylaxis and treatment of viral infection, for example by human immunodeficiency virus (HIV), which is believed to be the aetiological agent in human acquired immunodeficiency syndrome (AIDS).
There has been much interest in the use of nucleoside analogues as inhibitors of HIV. 2xe2x80x2,3xe2x80x2-dideoxy-2xe2x80x2,3xe2x80x2-didehydrothymidine (d4T) and 3xe2x80x2-azido-3xe2x80x2-deoxythymidine (AZT) are both known inhibitors of HIV [Hitchcock et al., Antiviral Chem. Chemother. (1991), 2, 125; Mansuri et al., Antimicrob. Agents Chemother., (1990), 34, 637.]. The inhibition of HIV by these, and other nucleoside analogues, is conventionally thought to depend upon conversion of the nucleoside analogue in vivo to the corresponding 5xe2x80x2-triphosphate by (host-cell) kinase enzymes. However, this absolute dependence upon (host-cell) kinase-mediated activation can lead to poor activity, the emergence of resistance, and clinical toxicity.
In order to reduce the dependence on kinase enzymes the use of masked phosphate pro-drugs of the bioactive nucleotide forms of several chemotherapeutic nucleoside analogues has been suggested [McGuigan et al., Nucleic Acids Res., (1989), 17, 6065; McGuigan et al., Ibid., (1989), 17, 7195; Chawla et al., J. Med. Chem., (1984), 27, 1733; Sergheraert et al., J. Med. Chem. (1993), 36, 826-830.]. In particular, McGuigan et al [J. Med. Chem. 36, 1048-1052 (1993)] have reported the preparation of aryl esterxe2x80x94phosphoramidate derivatives of AZT. In vitro evaluation of these compounds revealed the compounds to have anti-HIV activity. However, in xe2x80x9cnormalxe2x80x9d thymidine kinase rich (TK+) cells, the activity of such compounds was at least an order of magnitude less than the parent nucleoside AZT. Only in TK-deficient (TKxe2x88x92) cells, in which the activity of the aryl esterxe2x80x94phosphoramidate derivatives was virtually maintained but the activity of AZT was reduced, did the activity of the derivatives exceed that of AZT.
McGuigan et al [Bioorganic and Medical Chemistry Letters, 3,(6), 1203-1206 (1993)] have also reported preparation of triester phosphate derivatives of d4T. Again, in vitro evaluation of these compounds revealed that whilst the compounds have significant anti-HIV activity, the activity is less than that of the parent nucleoside d4T in TK+ cells.
Abraham and Wagner (Nucleosides and Nucleotides 13 (9). 1891-1903 (1994)) have reported the preparation of nucleoside phosphoramidate diesters and triesters but do not report any biological activity.
The acyclic nucleoside analogue 9(2-phosphonomethoxyethyly adenine (PMEA), and analogues thereof, have been demonstrated to show activity against herpes viruses and retroviruses including HIV (Calio et al., Antiviral Res., (1994), 23(1), 77-89; Balzarini et al., AIDS, (1991), 5(1), 21-28).
To date, the approach of providing masked phosphate pro-drugs has failed to enhance the anti-viral activities of the parent nucleoside analogues such as AZT and d4T in TK+ cells. Furthermore, the emergence of resistance to the nucleoside analogues in their bioactive 5xe2x80x2-triphosphate form has rendered the reported masked phosphate pro-drugs and their parent nucleoside analogues potentially ineffective.
It has now been found that a particular class of masked nucleoside analogues are highly potent viral inhibitors in both TKxe2x88x92 and TK+ cells, and yet retain activity against nucleoside (e.g. d4T)-resistant virus.
According to the present invention there is provided a compound of the formula (1) 
wherein Ar is an aryl group;
Y is oxygen or sulphur;
X1 is selected from O, NR3, S, CR3R4, CR3W1 and CW1W2 where R3 and R4 are independently selected from hydrogen, alkyl and aryl groups; and W1 and W2 are heteroatoms;
X2and Y6 together are direct bond; or X is CH2 and X2 is selected (independently of X1) from O, NR3, S, CR3R4, CR3W1 and CW1W2 where R3 and R4 are independently selected from hydrogen, alkyl and aryl groups; and W1 and W2 are heteroatoms;
X3 is a alkylene of 1 to 6 carbon atoms (e.g., alkenyl);
X4 is oxygen or CH2;
X5 may be absent or is CH2;
Z is selected from O, NR5, S, alkylene and arylene (e.g., alkenyl) groups, where R5 is selected from hydrogen, alkyl and aryl groups;
J is selected from hydrogen, alkyl, aryl, heterocyclic and polycyclic groups;
Q is selected from O, NR6, S, CR6R7, CR6W3 and CW3W4 where R6 and R7 are independently selected from hydrogen, alkyl and aryl groups; and W3 and W4 are heteroatoms;
T1 and T2 are independently selected from hydrogen and CH2R8, where R8 is selected from H, OH and F; or T1 and T2 are linked together and together are selected from the groups 
where R9 is selected from H, halogen, CN, NH2, CO-alkyl and alkyl; and R10, R11, R12 are independently selected from H, N3, halogen, CN, NH2, CO-alkyl and alkyl;
B is a purine or pyrimidine base;
or a pharmaceutically acceptable derivative or metabolite thereof.
The compounds of the present invention are potent anti-viral agents. In particular, they are highly active against HIV in both TKxe2x88x92 and TK+ cells. Particularly surprising is the activity of the compounds of the present invention against nucleoside-resistant HIV. These observations indicate that the activity of these compounds is not wholly dependent upon the conventional mode of action (requiring hydrolysis of the phosphate aryl ester and P-X1 bonds followed by kinase-dependent conversion to the 5xe2x80x2-triphosphate derivative), but arises from an entirely different mode of action. The experimental data presented herein indicates that the compounds and metabolites of the present invention are directly acting as reverse transcriptase (RT) inhibitors via a previously unrecognised metabolic pathway and mechanism of action.
Reference in the present specification to an alkyl group means a branched or unbranched, cyclic or acyclic, saturated or unsaturated (e.g. alkenyl or alkynyl) hydrocarbyl radical. Where cyclic, the alkyl group is preferably C3 to C12, more preferably C5 to C10, more preferably C5 to C7. Where acyclic, the alkyl group is preferably C1 to C16, more preferably C1 to C6, more preferably methyl. Reference in the present specification to alkoxy and aryloxy groups means alkyl-O- and aryl-O- groups, respectively. Reference to alkanoyl and aryloyl groups means alkyl-CO- and aryl-CO-, respectively.
Reference in the present specification to an aryl group means an aromatic group, such as phenyl or naphthyl, or a heteroaromatic group containing one or more, preferably one, heteroatom, such as pyridyl, pyrrolyl, furanyl and thiophenyl. Preferably, the aryl group comprises phenyl or substituted phenyl.
The alkyl and aryl groups may be substituted or unsubstituted, preferably unsubstituted. Where substituted, there will generally be 1 to 3 substituents present, preferably 1substituent. Substituents include halo, halomethyl (such as CF3 and CCl3), oxo, hydroxy, carboxy, carboxyalkyl, alkoxy, alkanoyl, alkanoyloxy, arloxy, arloyl, aryloyloxy, amino, alkylamino, dialkylamino, cyano, azide, nitro, thiol, alkylthiol, sulphonyl, sulphoxide, heterocyclic, alkyl, benzyl, and aryl (such as phenyl and substituted phenyl).
Reference in the present specification to heterocyclic groups means groups containing one or more, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, pyrrolidinyl, pyrrolinyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, tetrahydrofuranyl, pyranyl, pyronyl, pyridyl, pyrazinyl, pyridazinyl, piperidyl, piperazinyl, morpholinyl, benzofuranyl, isobenzofuryl, indolyl, oxyindolyl, isoindolyl, indazolyl, indolinyl, 7-azaindolyl, isoindazolyl, benzopyranyl, coumarinyl, isocoumarinyl, quinolyl, isoquinolyl, naphthridinyl, cinnolinyl, quinazolinyl, pyridopyridyl, benzoxazinyl, quinoxalinyl, chromenyl, chromanyl, isochromanyl and carbolinyl.
References in the present specification to polycyclic groups means a group comprising two or more non-aromatic carbcyclic or heterocyclic rings which may themselves be substituted.
Reference in the present specification to halogen means a fluorine, chlorine, bromine or iodine radical, preferably fluorine or chlorine radical.
The group Ar comprises a substituted or unsubstituted aryl group, wherein the term xe2x80x9caryl groupxe2x80x9d and the possible substitution of said group is as defined above. Preferably, Ar is a substituted or unsubstituted phenyl group. Particularly preferred substituents are election withdrawing groups such as halogen (preferably chlorine or fluorine), trihalomethyl (preferably trifluoromethyl), cyano and nitro groups. Preferably, Ar is phenyl, 3,5-dichloro-phenyl, p-trifluoromethyl-phenyl, p-cyano-phenyl, or p-nitro-phenyl.
Y may be oxygen or sulphur. Preferably, Y is oxygen.
X1 is from O, NR3, S, CR3R4, CR3W1 and CW1 W2 where R3 and R4 are independently selected from hydrogen, alkyl and aryl groups; and W1 and W2 are heteroatoms. Preferably, X1 is selected from O, S and NR3. Preferably, X1 is NR3. When present, R3 is preferably H. When present, W1 and W2 may independently comprise any heteroatom such as a halogen, preferably fluorine. fluorine
X2and X6 together are a direct bond; or X6 is CH2 and X2 is selected (independently of X1) from O, NR3, S, CR3R4, CR3W1 and CW1W2 where R3 and R4 are independently selected from H, alkyl and aryl groups; and W1 and W2 are heteroatoms. When present, X2 is preferably oxygen. When present, R3 is preferably H. When present W1 and W2 may independently comprise any heteroatom such as halogen, preferably fluorine.
X4 is oxygen or CH2. Preferably, X4 is oxygen.
X5 may be absent or is CH2.
Z may comprise O, NR5, S, alkyl e.g. alkenyl or aryl groups, where R5 is selected from H, alkyl and aryl groups. Preferably, Z is O or NR5. Preferably, R3 is hydrogen. Most preferably, Z is oxygen.
J is selected from hydrogen, alkyl, aryl, heterocyclic and polycyclic groups. Preferably, J is a substituted or unsubstituted alkyl group. Preferably, J is a substituted or unsubstituted C1-6 alkyl group, preferably a benzyl or methyl group. xe2x80x9cX3 is a C1-6 alkylene group. X3 may be a C1-6 substituted or unsubstituted, branched or unbranched, methylene chain.xe2x80x9d Preferably, X3 is a group CR1R2 where R1 and R2 are indepedently selected from hydrogen, alkyl and aryl groups. Preferably, at least one of R1 and R2 is hydrogen. It will be appreciated that if R1 and R2 are different, the carbon atom to which they are bonded is an asymmetric centre. The stereochemistry at this site may be R or S or mixed. When one of R1 and R2 is hydrogen, the stereochemistry is preferably S.
Q is selected from O, NR6, S, CR6R7, CR6W3 and CW3W4, where R6 and R7 are independently selected from hydrogen, alkyl and aryl groups; and W2 and W3 are heteroatoms such as halogen atoms, preferably fluorine. Preferably, Q is O, S, CH2 or CF2. Most preferably, Q is oxygen.
T1 and T2 are independently selected from hydrogen and CH2R4 where R8 is selected from H, OH and F; or T2 and T2 are linked together and together are selected from the groups: 
where R9 is selected from H, halogen, CN, NH2, CO-alkyl, and alkyl, preferably R9 is H or F; and R10, R11, and R12 are independently selected from H, N3, halogen, CN, NH2, CO-alkyl, and alkyl, preferably R10, R11 and R12 are independently selected from H, F and N3. It will be appreciated that R9 corresponds to the 3xe2x80x2-xcex1 position and R10 corresponds to the 3xe2x80x2-xcex2 position. Preferably, T1 and T2 are linked together and together form the group: 
B is a monovalent radical of unsubstituted thymine, cytosine, adenine, or guanine, or a monovalent radical of substituted uracil, thymine, cytosine, adenine, or guanine in which the substituents are as defined above; e.g., halo, halomethyl, oxo, hydroxy, carboxy, carboxyalkyl, alkoxy, alkanoyl, alkanoyloxy, arloxy, aryloyl, aryloyloxy, amino, alkylamino, dialkylamino, cyano, azide, nitro, thiol, alkylthiol, sulphonyl, sulphoxide, heterocyclic, alkyl, benzyl, and aryl. Preferably, B is adenine or thymine.
Preferably, the present invention provides a compound of formula (2) 
wherein Ar, R1, J, X2, X5, X6, Q, T1, T2 and B are as defined above; or a pharmaceutically acceptable derivative or metabolite thereof.
It will be appreciated that the group xe2x80x94NHxe2x80x94CHR1xe2x80x94CO2J corresponds to a carboxy-protected xcex1-amino acid. Preferably, the group R1 corresponds to the side chain of a naturally occurring amino acid such as alanine (R1 is methyl), arginine (R1 is 3-guanidinopropyl), asparagine (R1 is carbamoylethyl), asparatic acid (R1 is carboxymethyl), cysteine (R1 is mercaptomethyl), cystine 9R1 is 2-amino-2-carboxyethyldithiomethyl), glycine (R1 is hydrogen), glutamic acid (R1 is 2-carboxyethyl), glutamine (R1 is 2-carbamoylethyl), hystidine (R1 is 1H-imidazolemethyl), isoleucine (R1 is but-2-yl), leucine (R1 is butyl), lysine (R1 is 4-aminobutyl), methionine (R1 is 2-methylthioethyl), phenylalanine (R1 is benzyl), serine (R1 is hydroxymethyl), threonine (R1 is 1-hydroxyethyl), tryptophan (r1 is 1H-indol-3-ylemthyl), tyrosine (R1 is 4-hydroxybenzyl), and valine (R1 is prop-2-yl). Preferably R1 is Me or PhCh2 corresponding to the side chain of alanine or phenylalanine, respectively. Preferably, the stereochemistry at the asymmetric center xe2x80x94CHR1xe2x80x94 corresponds to an L-amino acid.
According to one preferred embodiment, the present invention provides a compound of formula (3): 
wherein Ar, Y, X1, X2, X3, X4, Z, Q and B are as defined above.
More preferably, the invention provides a compound, according to formula (3), of formula (4): 
wherein Ar, R1 and J are as defined above; or a pharmaceutically acceptable derivative or metabolite thereof. Preferably, the invention provides a compound of formula (4) in which Ar, R1 and J are defined in accordance with Table 1.
According to a further preferred embodiment, the present invention provides a compound of formula (5) 
wherein Ar, Y, X1, X2, X3, X4, Z, J, R9, R10, R11, R12, Q and B as defined above.
More preferably, the invention provides a compound, according to formula (5), of the formula (6): 
wherein Ar, R1, J, R9, R10, R11, R12, and B are as defined above.
According further preferred embodiment, the present invention provides a compound of formula (7): 
wherein Ar, Y, X1, X3, X4, Z, J, Q and B are as defined above and T1 and T2 are independently selected from H and CH2R8 wherein R8 is as defined above. Preferably, B is a purine base. More preferably, B is adenine. Preferably, T1 is hydrogen. Preferably, T2 is CH2R8. These compounds are analogues of the acyclic nucleoside analogue 9-(2-phosphonylmethoxyethyl) adenine (PMEA), which has been demonstrated to show activity against herpes viruses and retroviruses (Calio et al., Antiviral Res., (1994), 23(1), 77-89; Balzarini et al., AIDS, (1991), 5(1), 21-28).
More preferably, the invention provides a compound, according to formula (7), of formula (8): 
wherein Ar, R1, J, T1, T2 and B are as defined above.
It is a feature of the aryl ester phosphate compounds (1) of the present invention that they exhibit significantly enhanced anti-viral efficacy, in both in vitro and in vivo tests, in comparison to their corresponding nucleoside analogue (9) 
In addition, the compounds of the present invention exhibit significantly reduced toxicity in comparison to their corresponding analogue (9).
The compounds of the present invention thus exhibit a greatly enhanced selectivity index (ratio of CC50 (toxicity):EC50 (activity)) in comparison to their corresponding nucleoside analogue.
Experiments with radiolabelled compounds of the present invention have shown that the compounds give enhanced intracellular levels of nucleoside 5xe2x80x2-triphosphate, the enhancement being particularly significant in TKxe2x88x92 cells. Thus, the compounds of the present invention may act in part by the known metabolic pathway.
However, it has been found that the compounds of the present invention show surprising activity against nucleoside resistant strains of HIV. This indicates that the compounds of the present invention are also acting by a pathway independent of a 5xe2x80x2-triphosphate metabolite.
It has been demonstrated that the compounds of the present invention lead to intracellular generation of high levels of a metabolite (10). 
Metabolite (10) may also be prepared by treatment of the corresponding compound according to formula (1) with hog liver esterase. Moreover, it has been shown that compounds of formula (10) are direct inhibitors of reverse transcriptase from HIV.
According to a further aspect of the present invention there is provided a compound of formula (10) 
wherein Ar, Y, X1, X2, X3, X4, X6, T1, T2, Q, X5 and B are as defined above, or a pharmaceutically acceptable derivative or metabolite thereof.
The intracellular generation of anti-viral metabolites such as (10) is an important feature of the invention for several reasons. Firstly, the direct activity of (10) on RT removes the necessity for further nucleotide-kinase mediated phosphorylation, which may be slow in many cases. In cases where the nucleoside monophosphate is not a substrate for host nucleotide kinases, activation will be poor and anti-viral efficacy low, even if the triphosphate is an excellent RT inhibitor. In such cases, the generation of metabolites such as (10) may lead to a very significant enhancement in antiviral action. Such compounds may be acting directly in their own right or via a rearrangement, decomposition or disproportionation product or via a contaminant. Moreover, the structure of metabolites such as (10) may be further designed to optimise binding to the known structure of RT, and such modified metabolites could be delivered intracellularly using technology herein described, to further enhance the anti-viral effect.
By xe2x80x9ca pharmaceutically acceptable derivativexe2x80x9d is meant any pharmaceutically acceptable salt, ester or salt of such ester or any other compound which upon administration to a recipient is capable of providing (directly or indirectly) a compound of formula (1) or (10). By xe2x80x9cpharmaceutically acceptable metabolitexe2x80x9d is meant a metabolite or residue of a compound of formula (1) or (10) which gives rise to a nucleoside-resistance independent or nucleoside 5xe2x80x2-triphosphate independent mode of reverse transcriptase inhibition exhibited by the compounds of formula (1) or (10).
According to a further aspect of the present invention there is provided a compound according to the present invention for use in a method of treatment, preferably in the prophylaxis or treatment of viral infection.
According to a further aspect of the present invention there is provided use of a compound according to the present invention in the manufacture of a medicament for the prophylaxis or treatment of viral infection.
According to a further aspect of the present invention there is provided a method of prophylaxis or treatment of viral infection comprising administration to a patient in need of such treatment an effective dose of a compound according to the present invention.
The viral infection may comprise any viral infection such as HIV and herpes virus, including HSV 1 and HSV 2, CMV, VZV, EBV, HAV, HBV, HCV, HDV, papilloma, rabies and influenza.
Preferably, the viral infection comprises HIV infection, more preferably HIV-I or HIV-II. It is a feature of the present invention that the compounds exhibit good activity against both HIV-I and HIV-II.
According to a further aspect of the present invention there is provided use of a compound of the present invention in the manufacture of a medicament for use in the inhibition of a reverse transcriptase by a nucleoside-resistance independent or nucleoside 5xe2x80x2-triphosphate independent mode of action.
According to a further aspect of the present invention there is provided a pharmaceutical composition comprising a compound of the present invention in combination with a pharmaceutically acceptable excipient.
According to a further aspect of the present invention there is provided a method of preparing a pharmaceutical composition comprising the step of continuing a compound of of the present invention with a pharmaceutically acceptable excipient.
The medicaments employed in the present invention can be administered by oral or parenteral routes, including intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal, airway (aerosol), rectal, vaginal and topical (including buccal and sublingual) administration.
For oral administration, the compounds of the invention will generally be provided in the form of tablets or capsules, as a powder or granules, or as an aqueous solution or suspension.
Tablets for oral use may include the active ingredient mixed with pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavouring agents, colouring agents and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrating agents. Binding agents may include starch and gelatin, while the lubricating agent, if present, will generally be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract.
Capsules for oral use include hard gelatin capsules in which the active ingredient is mixed with a solid diluent, and soft gelatin capsules wherein the active ingredient is mixed with water or an oil such as peanut oil, liquid paraffin or olive oil.
Formulations for rectal administration may be presented as a suppository with a suitable base comprising for example cocoa butter or a salicylate.
Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the active ingredient such carriers as are known in the art to be appropriate.
For intramuscular, intraperitoneal, subcutaneous and intravenous use, the compounds of the invention will generally be provided in sterile aqueous solutions or suspensions, buffered to an appropriate pH and isotonicity. Suitable aqueous vehicles include Ringer""s solution and isotonic sodium chloride. Aqueous suspensions according to the invention may include suspending agents such as cellulose derivatives, sodium alginate, polyvinyl-pyrrolidone and gum tragacanth, and a wetting agent such as lecithin. Suitable preservatives for aqueous suspensions include ethyl and n-propyl p-hydroxybenzoate.
The compounds of the invention may also be presented as liposome formulations.
In general a suitable dose will be in the range of 0.1 to 300 mg per kilogram body weight-of the recipient per day, preferably in the range of 6 to 150 mg per kilogram body weight per day and most preferably in the range 15 to 100 mg per kilogram body weight per day. The desired dose is preferably presented as two, three, four, five or six or more sub-doses administered at appropriate intervals throughout the day. These sub-doses may be administered in unit dosage forms, for example, containing 10 to 1500 mg, preferably 20 to 1000 mg, and most preferably 50 to 700 mg of active ingredient per unit dosage form.
According to a further aspect of the present invention there is provided a process for the preparation of a compound according to the present invention, the process comprising reaction of a compound of formula (11) 
with a compound formula (12) 
The reaction may be carried out in the tetrahydrofuran in the presence of N-methylimidazole.
Alternatively, the compounds of the present invention may be prepared by reaction of a compound of formula (13) or a suitable derivative thereof 
with ArOH and a compound of formula (14) or suitable derivatives thereof 